Hemophilia A is a severe bleeding disorder, resulting from a deficiency of coagulation Factor VIII (FVIII). A candidate therapeutic approach for this disease is gene therapy aimed to provide constant delivery of FVIII into the systemic circulation by means of in vivo genetic modification. While adenovirus 5-based vectors have proven promising as FVIII gene delivery vehicles, the previous vector generations suffer from size limitations which prevent accommodation of full-length FVIII cDNA, and from toxicity due to viral gene expression in the transduced cells. A novel minimal adenovirus vector, the Mini-AdFVIII, devoid of all viral genes and retaining minimal cis-elements of the viral genome, that carries the full- length human FVIII cDNA under control of the human albumin promoter eliminates these problems. Tail vein administration of Mini-AdFVIII resulted in efficient transduction of biologically active human FVIII in plasma. The studies proposed extend these findings to assess the potential benefit of multiple administrations of the Mini-AdFVIII resulted in efficient transduction of biologically active human FVIII in plasma. The studies proposed extend these findings to assess the potential benefit of multiple administrations of the Mini-AdFVIII vector in terms of increased Factor VIII expression. The immune responses of the multiple treated mice and the status of the Factor VIII transgene (integrated vs. episomal) will be measured. PROPOSED COMMERCIAL APPLICATIONS: Hemophilia A is the most common inherited severe bleeding disorder, characterized by a deficiency in coagulation Factor VIII affecting approximately 1 in 10,000 males. Approximately 50,000 patients have hemophilia A worldwide (U.S. and Europe) with half of these patients requiring repeated injections of FVIII at a cost of $80,000 per patient per year. A long lasting genetic therapy would reduce cost, discomfort, inconvenience, and disease complications from fluctuations in FVIII levels associated with injections.